1. Field of the Invention
The present invention relates generally to the delivery of gene medicines to the brain. More particularly the present invention involves the combination of liposome technology, blood-brain barrier (BBB) receptor technology, pegylation technology, and therapeutic gene technology to provide formulations which are useful in the non-invasive delivery of genes to the brain.
2. Description of Related Art
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and grouped in the appended bibliography.
The expression of exogenously administering genes in brain has previously been achieved in vivo with either viral vectors or cationic liposomes (1-4). However, in either case, highly invasive routes of administration are required. Such invasive techniques are needed because of the failure of either viruses or cationic liposomes to cross the brain capillary wall, which forms the blood-brain barrier (BBB) in vivo. The existence of the BBB necessitates the administration of the exogenous gene either intracerebrally via craniotomy (1), or by the intra-carotid arterial infusion of noxious agents that cause BBB disruption and transient opening of the BBB (4).
Human gene therapy of the brain will likely require repeated administration of the gene medicine. Therefore, it would be advantageous to administer the gene by a route that is no more invasive than a simple intravenous injection. With this approach, the gene therapeutic is delivered through the BBB by targeting the gene medicine to the brain via endogenous BBB transport systems (5). Carrier-mediated transport (CMT) systems exist for the transport of nutrients across the BBB (5). Similarly, receptor-mediated transcytosis (RMT) systems operate to transport circulating peptides across the BBB, such as insulin, transferrin, or insulin-like growth factors (5). These endogenous peptides can act as xe2x80x9ctransporting peptides,xe2x80x9d or xe2x80x9cmolecular Trojan horses,xe2x80x9d to ferry drugs across the BBB. In this approach, called the chimeric peptide technology, the drug that is normally not transported across the BBB is conjugated to a xe2x80x9ctransportable peptide, and the drug/transportable peptide conjugate undergoes RMT through the BBB (U.S. Pat. No. 4,801,575).
Peptidomimetic monoclonal antibodies (MAb) that bind endogenous transport systems within the BBB, such as the transferrin receptor (TfR) or insulin receptor, have been used in previous studies for targeting neuropeptides or antisense agents through the BBB in vivo (5). The ability of certain receptor-binding MAbs to mimic the action of the endogenous peptide that binds the same receptor is well known in the literature (35-37). In addition, the ability of such peptidomimetic MAbs, such as anti-TfR MAbs, to transport drugs into cells via these receptor-mediated endocytosis is also well known (38).
The expression in the brain of a therapeutic gene requires that the gene formulation that is injected into the blood is transported not only across the BBB by RMT, but also across the brain cell membrane (BCM) by receptor-mediated endocytosis (RME) into the target cell in brain. In addition, using endogenous BBB transport systems to target gene medicines non-invasively to the brain also requires the development of a suitable formulation of the gene therapeutic that is stable in the bloodstream. Cationic liposome/DNA complexes have been used for in vivo gene expression, but these formulations aggregate extensively in saline solution (6-11). This aggregation results in selective gene expression in the lung with little expression in peripheral tissues (12-14), and no expression in brain following intravenous administration of the cationic liposome/DNA complex (12). The DNA plasmid could be conjugated to the peptidomimetic MAb via a cationic polylysine bridge (15-17). However, electrostatic interactions between DNA and polycations may not be stable in blood, and highly polycationic proteins such as histone or polylysine exert toxic effects at the BBB and cause generalized BBB permeability changes in vivo (18).
In accordance with the present invention, therapeutic genes are introduced non-invasively into the brain across the blood brain barrier. Once inside the brain, the therapeutic genes express therapeutic agents which are useful in the diagnosis and treatment of brain disease. The present invention is based on the use of liposomes which are capable of delivering therapeutic genes across the blood-brain barrier.
The liposomes of the present invention include a neutral liposome having an exterior surface and an internal compartment in which the therapeutic gene is located. The surface of the liposome is decorated with several thousand strands of polyethyleneglycol (PEG), a process called xe2x80x9cpegylation.xe2x80x9d The PEG strands make the surface of the liposome xe2x80x9chairy,xe2x80x9d and this prevents the rapid absorption of blood proteins to the surface of the liposome, which is what accelerates the rapid removal from blood of unprotected liposomes. In contrast, the pegylated liposomes are protected and are removed from blood at a much slower rate. The PEG strands also act as xe2x80x9cconjugation agentsxe2x80x9d for attachment of the xe2x80x9ctransportable peptidexe2x80x9d to the surface of the pegylated liposome, which is what triggers the RMT of the complex through the BBB and the RME through the BCM in vivo. The therapeutic gene includes a sufficient amount of DNA to encode a therapeutic agent. A plurality of blood-brain barrier targeting agents are attached to the liposome surface via a conjugation agent. The therapeutic gene located within the immunoliposome targeting vehicle is transported across the blood-brain barrier and released into the interstitial space of brain. Once there, the xe2x80x9cpegylated liposomexe2x80x9d undergoes receptor-mediated endocytosis into target cells in brain because the surface of the liposome is decorated with xe2x80x9ctransportable peptidesxe2x80x9d that recognize receptor located on the brain cell plasma membrane (BCM). Owing to the presence of insulin or transferrin receptors on both the BBB and the BCM, the xe2x80x9ctransportable peptidexe2x80x9d catalyzes transport across both of the 2 barriers in brain: the BBB and the BCM. Once inside the target brain cell, the liposome complex is entrapped within brain cell endosomes, followed by release of the gene therapeutic into the ctoplasm of brain cells, where it can enter the nucleus, resulting in expression of the therapeutic agent.
It was found in accordance with the present invention that the use of liposomes in which the polyethyleneglycol is conjugated to the liposome surface results in an increase in the plasma bioavailability of the DNA incorporated within the interior of the immunoliposome. It was also found that the stability of the DNA located within the immunoliposome is increased during in vivo use. Further, in addition to achieving expression of an exogenous gene in the brain, it is also possible to achieve, in parallel, gene expression in other organs which contain or express high levels of the receptor targeted by the blood-brain barrier targeting agent.
The above described and many other features and attendant advantages of the present invention will become better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.